Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMPmediated, protein kinase A-dependent local subsarcolemmal ryanodine receptor Ca2+ releases (LCRs). LCRs activate an inward Na+/Ca2+ exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase A-dependent phospholamban phosphorylation, and increased SANC firing rate by 55%, indicating a high basal activity of PDEs in SANCs. cAMP-degrading PDE1, PDE3 and PDE4 represent major PDE activities in rabbit SANC, and PDE inhibition by IBMX increases spontaneous firing of SANC by 50%. Though inhibition of single PDE1-PDE4 only moderately increases spontaneous SANC firing, dual PDE3+PDE4 inhibition produces synergistic effect hastening spontaneous SANC beating rate by 50%. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, i.e., PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca2+ release, Na+/Ca2+ exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine, neither IBMX nor dual PDE3+PDE4 inhibition was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibition-induced augmentation of ICa,L amplitude. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMPmediated, protein kinase A-dependent increase of diastolic ryanodine receptor Ca2+ release and restrict the spontaneous SANC beating rate. To study how PDE inhibition controls the SR Ca2+ refilling and LCR period, we compared kinetics of SR Ca2+ refilling in control and after PDE inhibition. Phosphorylation of phospholamban (PLB) has been used as index of SR pumping rate and SR refilling was estimated by the time to 90% decay of the AP-initiated global cytosolic Ca2+ transient (T-90). Graded PLB phosphorylation by a broad-spectrum phosphodiesterase inhibitor (IBMX), by selective PDE3 or PDE4 inhibition or specific PKA inhibitor peptide (PKI) were paralleled by proportional changes in T-90. Concomitant changes in T-90 and LCR period were highly correlated with changes in the spontaneous cycle length. Similar to intact SANC, inhibition of PDE3 alone with 0.3 mkM cilostomide (n=8) or PDE4 alone with 2 mkM rolipram (n=7) had minor effect on LCR number or size in permeabilized SANC. In contrast, dual inhibition of PDE3 and PDE4 produced marked increase in LCR number (n=6) and size (n=6), and caffeine- induced SR Ca2+ content in permeabilized SANC (*P < 0.05). Expression of PDE1A protein was 5-fold higher in rabbit sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in rabbit SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact SANC PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging) but located approximately within about 150 nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca2+ cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). Results obtained in isolated rabbit SANC were recently confirmed in the mouse isolated sinoatrial node (SAN). Suppression of PDE activity by the broad spectrum PDE inhibitor IBMX (100 micro mol/L) increased the spontaneous beating rate of mouse SAN from 375bpm to 623 bpm (n=6, P<0.05). The positive chronotropic effects of IBMX indicate that an increase in intracellular cAMP level activates PKA and accelerates SAN automaticity, independent of external receptor stimulation. Stated in other terms, these data indicate constitutively active PDE in mouse SAN tissue, which degrades cAMP, leads to suppression of downstream cAMP-mediated PKA-dependent signaling. When PDE activity is blunted, PKA dependent signaling and the spontaneous beating rate are markedly enhanced in mouse SAN. This idea was tested using anti-PS16 PLB antibody and PLB total antibody. On average, IBMX increased the ratio of PS-16 PLB to total PLB by 51% comparing to its basal level (n=10 cells, P<0.05). Like in isolated rabbit SANC, PDE inhibition in mouse SANC increased LCR occurrence, size and duration. In permeabilized mouse SANC, in conditions of tight control of cytoplasmic Ca2+, IBMX significantly (p<0.03) increased LCRs size (from 4.4 to 6.4 mkm), duration (38 to 56 msec), the Ca2+ signal of LCR ensemble (more than 2 fold), the percent of cells that produce periodic LCRs (from 72% in control to 100 % during IBMX application), the rhythmicity index (RI, assessed via autocorrelation function). Thus, suppression of PDE activity increases cAMP-mediated PKA-dependent phosphorylation, accelerates the SR Ca2+ refilling rate, decreases the LCR period, stimulates the Ca2+ clock, and increases the spontaneous beating rate of isolated SANC and SAN tissue. Similar to isolated rabbit and mouse SANC, PDE inhibition with IBMX in permeabilized rabbit ventricular myocytes (VM) increased the number of LCR events and LCR periodicity measured by Fast Fourier Transforms (FFT), and clock-like local Ca2+ releases, partially synchronized in space and time, emerged, similar to pacemaker cells. This ensemble of rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. When we measured RyR2 phosphorylation by phospho-imaging of permeabilized VM, we found significant enhancement of RyR2 phosphorylation at Ser2809 in response to PDE inhibition by IBMX compared to control. Addition of IBMX in the presence of the protein phosphatase (PP) inhibitor CyA further enhanced PKA-dependent phosphorylation, which resulted in further increase of the amplitude, width, duration and number of LCRs, which not only further amplified the Ca2+ signal of individual LCRs but also amplified the Ca2+ signal of the LCR ensemble by 8-fold over control in permeabilized ventricular myocytes (VM). Thus, suppression of PDE activity in VM has a similar effect on the Ca2+ clock as in pacemaker cells. Specifically, it accelerates the SR Ca2+ cycling via phosphorylationdependent increase in the rate of SR Ca2+ pumping or kinetics of RyR activation, or by the reduction in the threshold for spontaneous RyR activation. Conclusion: Spontaneous LCRs in rabbit SANC are regulated by dual PDE3 and PDE4 activation. Inhibition of PDE3 alone (with 0.3 to 0.9 mkM cilostomide) or PDE4 alone (with 2 to 6 mkM rolipram), or dual inhibition of both PDEs in freshly isolated rabbit SANC did not cause significant changes in the total cAMP concentration in the cells, opposite to the total PDE inhibition by IBMX (100 mkM); these data can be explained by the hypothesis that local changes in cAMP caused by PDE3 and PDE4 inhibition (what is reflected in our functional data) cannot be detected via this method of tot